1. Field of the Invention
The present invention relates to repressor proteins that: (i) recognize the lactose operator with increased affinity compared to wild type protein and exhibit normal inducibility; (ii) recognize the lactose operator with normal affinity while exhibiting enhanced inducibility (i.e., increased sensitivity to normal inducing sugars or responsiveness to alternative ligands); or (iii) alternatively, repressor proteins exhibiting increased affinity for the lactose operator DNA and enhanced inducibility.
2. Description of Related Art
The lac repressor protein is a genetic regulatory protein used widely to control the expression of cloned genes and is the prototype for negative control of transcription initiation in E. coli (Jacob & Monod, 1961). The lac repressor normally regulates expression of the lactose metabolic enzymes and couples cellular response with environmental availability of metabolites (Miller & Reznikoff, 1980). Multiple vector systems are commercially available that employ this repressor protein in cloning genes and overexpressing their protein products. These systems rely on the ability of the lac repressor to inhibit transcriptional initiation in the absence of an inducer.
FIG. 1 shows a schematic depicting how the lac repressor protein (LacI) and lac operator (LacO) work. The “i” gene product forms a tetrameric lactose repressor protein (LacI), which binds with high affinity to the partially two-fold symmetric lac operator DNA target sequence (LacO) that overlaps the promoter sequence “p”. RNA polymerase binding, transcriptional initiation, and/or transcriptional elongation are inhibited when Lac1 occupies this site, thereby precluding production of the mRNA encoding the lac enzymes (z, y, a) (reviewed in Matthews & Nichols, 1998). In the absence of inducer sugars, the high affinity of LacI for LacO allows production of only small quantities of lacZYA mRNA. However, when inducer sugars, “I”, are present, they bind LacI and cause a conformational change (depicted as (O→□)) (Lewis et al., 1996), which reduces the repressor's LacO binding affinity without having effect on its binding affinity for nonspecific DNA (Lin & Riggs, 1975). Excess non-LacO DNA in the cell thereby effectively competes for binding to the protein and sequesters the repressor-inducer complex, allowing transcription of downstream lac mRNA to proceed for as long as inducer sugar is available. When inducer sugar levels are depleted, inducer dissociates from the repressor protein. The repressor protein subsequently reassumes the conformation with high affinity for LacO DNA, associates with LacO, and shuts down further synthesis of lac mRNA. The lactose regulatory cycle, therefore, involves association with both specific and non-specific DNA sequences, binding of inducer sugar molecules (ligands), and conformational shifts in response to these ligands.
When lactose is available in the environment, the low constitutive amounts of lac permease transport this sugar into the cell, and the correspondingly low levels of β-galactosidase result in production of the natural in vivo inducer, β-1,6-allolactose (Jobe & Bourgeois, 1972). When lactose levels are sufficiently decreased, the intracellular store of β-1,6-allolactose is depleted by β-galactosidase hydrolysis. These enzymatic activities ensure that the lac enzymes are not expressed except in the presence of lactose. In the E. coli genome, two additional operator sequences, located within the “i” and “z” genes, bind LacI with lower affinity (Miller & Reznikoff, 1980). Each dimer of the LacI tetramer binds one operator sequence; simultaneous binding of tetrameric LacI to two operators results in looped DNA and enhances repression (reviewed in Matthews & Nichols, 1998).
A different inducer, isopropyl-β,D-thiogalactoside (IPTG), that is not hydrolyzed by β-galactosidase, is commonly used to turn on transcription of genes cloned under control of the lac promoter-operator (in place of the z, y, and a genes depicted in FIG. 1). For expression systems that employ LacI/LacO interaction, the secondary operator sequences are usually not present, and the level of repression is consequently diminished.
For proteins that exert toxic effects or for which expression prior to addition of inducing sugar is otherwise deleterious, the ability to increase the efficacy of LacI repression while still maintaining a normal induction response would be very useful. Different, unique repressor proteins that recognize the primary lac operator sequence (O1) with increased affinity, yet exhibit binding comparable to wild-type LacI in the presence of inducer (i.e., exhibit a normal induction response) would significantly enhance expression for many genes. Such repressor proteins could be used in any system in which the lac operator is used as the target sequence for transcriptional control and would provide an enhanced regulatory mechanism for the expression of cloned protein products.